JP5564735B2 - Aquaporin 3 expression regulator - Google Patents

Description

  The present invention relates to an aquaporin 3 expression regulator (expression enhancer and expression inhibitor) and use thereof.

  Aquaporin (hereinafter abbreviated as AQP) is a 6-membrane transmembrane protein discovered by Agre in 1992, and is an important protein that functions as a water channel that regulates the amount of water in cells. Aquaporins exist universally from bacteria to mammals, and so far, 13 types of aquaporins from AQP0 to AQP12 have been confirmed in mammals.

  Aquaporin 3 (AQP3), a type of water channel, is abundant in skin keratinocytes and is important for physiological moisturization of the skin by promoting the transport of water and glycerol. In addition, according to the study by the present inventors, the expression of AQP3 is markedly reduced at the site of skin inflammation, suggesting that this is closely related to the formation of dry symptoms associated with various skin diseases. Yes. Furthermore, AQP3 is also involved in the ability of keratinocytes to proliferate and migrate, and this channel is involved in promoting healing during wounding, while it is involved in malignant transformation in skin cancer. That is, substances that promote the expression of AQP3 are useful as cosmetics and pharmaceuticals having a moisturizing effect and wound healing effect, and substances that suppress the expression of AQP3 can be expected to be applied as antitumor agents.

  Central Asian plant Ajuga turkestanica (J Drugs Dermatol. 2007, 6 (6 Suppl): s20-4.) Has been found as a material that increases aquaporin 3 and shows a moisturizing effect. Has been put to practical use. In addition, retinoic acid has been reported to have an effect of promoting the expression of aquaporin 3 (Cao et al., J cell Physiol, 215, 506, 2008). However, there are very few aquaporin 3 expression regulators that may be put to practical use as cosmetics or pharmaceuticals.

  On the other hand, it has been reported that mussel has a moisturizing effect and can be used as a cosmetic material (JP-A-6-92838, JP-A-9-2935, JP-A-9-30931). However, the mechanism of action by which mussels exhibit a moisturizing effect is unknown. Moreover, it is described in international publication WO03 / 015808 that the water-soluble extract of several crude drugs containing a mussel shows a wound healing effect.

J Drugs Dermatol. 2007, 6 (6 Suppl): s20-4. Cao et al., J cell Physiol, 215, 506, 2008

Japanese Patent Laid-Open No. 6-92838 Japanese Patent Laid-Open No. 9-2935 Japanese Patent Laid-Open No. 9-30931 International Publication WO03 / 015808

  An object of the present invention is to provide an aquaporin 3 expression enhancer and an expression inhibitor useful for the prevention and / or treatment of a pathological condition caused by an abnormal function of aquaporin 3.

  The present inventors searched for AQP3 expression regulators in an in vitro experimental system, and the methanol extract of herbal medicinal mussel showed a significant AQP3 expression promoting action, and the methanol extract of ginger showed an AQP3 expression inhibitory action. Found to show. In addition, it was confirmed that the methanol extract of herbal medicine mussel shows a wound healing promoting action that is considered to be based on the promotion of AQP3 expression. The present invention has been completed based on these findings.

That is, according to the present invention, there is provided an aquaporin 3 expression enhancer containing a mussel extract as an active ingredient.
Preferably, the mussel extract is a mussel organic solvent extract.
Preferably, the organic solvent is a lower alcohol.
Preferably, the organic solvent is methanol.

Preferably, the aquaporin 3 expression enhancer of the present invention is used as a skin moisturizer.
Preferably, the aquaporin 3 expression enhancer of the present invention is used as a cosmetic.
Preferably, the aquaporin 3 expression enhancer of the present invention is used as a keratinocyte migration promoter.
Preferably, the aquaporin 3 expression enhancer of the present invention is used as a wound healing promoter.

According to the present invention, there is further provided an expression inhibitor of aquaporin 3 comprising a pepper extract as an active ingredient.
Preferably, the show extract is a show organic solvent extract.
Preferably, the organic solvent is a lower alcohol.
Preferably, the organic solvent is methanol.

The present invention further provides a method for enhancing aquaporin 3 expression comprising administering a mussel extract in vitro or in vivo.
The present invention further provides a method for suppressing the expression of aquaporin 3, which comprises administering a show extract in vitro or in vivo.

The present invention further provides use of a mussel extract for producing an aquaporin 3 expression enhancer.
According to the present invention, there is further provided use of a ginger extract for the production of an aquaporin 3 expression inhibitor.

  Aquaporin 3 that is abundant in keratinocytes is known to have both a skin moisturizing action and a wound healing promoting action. In the present invention, it has been demonstrated that mussel extract exhibits an aquaporin 3 expression promoting action and a keratinocyte migration promoting action, and thus exhibits a wound healing promoting action. The aquaporin 3 expression enhancer comprising a mussel extract according to the present invention as an active ingredient is useful as a skin moisturizer or wound healing promoter, specifically, a medicinal product as a wound healing agent for the purpose of promoting wound healing, It can be used as a cosmetic for improving rough skin or dry skin, and as a health food. Furthermore, in the present invention, it was demonstrated that the ginger extract exhibits an aquaporin 3 expression inhibitory action. The aquaporin 3 expression inhibitor containing the salmon extract according to the present invention as an active ingredient can be applied as an antitumor agent, for example.

FIG. 1 shows the action of various herbal extracts on aquaporin 3 mRNA expression in keratinocytes. FIG. 2 shows the action of various herbal extracts on aquaporin 3 protein expression in keratinocytes. FIG. 3 shows the effect of mussel on the migration ability of keratinocytes. FIG. 4 shows the reduction of aquaporin 3 mRNA expression at the site of skin inflammation. FIG. 5 shows the action of each fraction prepared from mussel extract on aquaporin 3 mRNA expression.

Hereinafter, embodiments of the present invention will be described in detail.
The present invention relates to (1) an aquaporin 3 expression enhancer containing a mussel extract as an active ingredient, and (2) an aquaporin 3 expression inhibitor containing a pepper extract as an active ingredient.

  Since the mussel extract has an action of enhancing the expression of aquaporin 3, it is useful as an active ingredient of a medicament for the prevention and / or treatment of a pathological condition caused by an abnormal function of aquaporin 3. Abnormal function of aquaporin 3 refers to the state of aquaporin 3 in a tissue cell under pathological conditions different from the state of aquaporin 3 in cells in a healthy tissue, a decrease in aquaporin 3 function, the number of aquaporin 3 in the cell Including reduction. The aquaporin 3 expression enhancer of the present invention containing a mussel extract as an active ingredient can be specifically used as a skin moisturizing agent or a wound healing promoter, but is not limited to these uses only. It can be administered to humans or animals as a cosmetic or pharmaceutical composition for the purpose of preventing or treating diseases of tissues or organs that express aquaporin 3.

  Since the ginger extract has an action of suppressing the expression of aquaporin 3, it is useful as an active ingredient of a medicament for the prevention and / or treatment of a pathological condition caused by an abnormal function of aquaporin 3. Abnormal function of aquaporin 3 refers to the state of aquaporin 3 in a tissue cell under a pathological condition different from the state of aquaporin 3 in cells in a healthy tissue, the increase in aquaporin 3 function, the number of aquaporin 3 in the cell Including an increase. The aquaporin 3 expression-enhancing agent of the present invention containing a salmon extract as an active ingredient can be specifically used as an anti-tumor agent, but is not limited to these applications, and is not limited to a tissue expressing aquaporin 3. It can be administered to humans or animals as cosmetic and pharmaceutical compositions intended for the prevention and treatment of organ diseases.

The mussel extract used in the present invention is a component extracted from a mussel (Schizonepeta tenuifolia Briquet (var. Japonica Kitagawa)) (flower ear or whole plant) belonging to the family Lamiaceae.
The ginger extract used in the present invention is a component extracted from the rhizome of Zingiberaceae ginger (Zingiber officinale Roscoe).

  Although the extraction method is not particularly limited, a preferable method is a method of first extracting the above-mentioned plant material appropriately cut or pulverized from room temperature under heating. The solvent used for extraction is preferably an organic solvent, such as lower alcohol (methanol, ethanol, propyl alcohol, isopropyl alcohol, etc.), polyhydric alcohol (glycerin, propylene glycol, etc.), lower alkyl ester (ethyl acetate, etc.), ketone (Acetone, methyl ethyl ketone, etc.), hydrocarbons (benzene, hexane, etc.), ethers (diethyl ether, etc.) and the like can be mentioned. Said solvent may be used independently and may mix and use two or more types of solvents. Among the above, lower alcohols are preferable, and methanol is particularly preferable.

  In addition, in the international publication WO03 / 015808, it is described that the herbal medicine is prepared as a water-soluble extract. However, the action of promoting the aquaporin 3 expression of mussels found in the present invention is a methanol extract, Among them, it is recognized in the 100% methanol elution fraction having relatively high hydrophobicity. Therefore, the enhanced expression of aquaporin 3 according to the present invention is considered to be an effect derived from a component different from that of International Publication No. WO03 / 015808.

  As extraction conditions, the extraction solvent is added in an amount of about 2 to 100 times, preferably 3 to 30 times the amount of the raw material, followed by extraction with stirring at room temperature or for several hours to several days as necessary. Is preferred. An extraction liquid can be obtained by filtering and removing an extraction residue after extraction. The obtained extract may be subjected to treatments such as dilution, concentration, drying, and purification according to a conventional method, if necessary.

  As the aquaporin 3 expression enhancer and the aquaporin 3 expression enhancer of the present invention, a mussel extract or a ginger extract may be used as it is, or a formulation of the extract may be used. The mussel extract or ginger extract is used in any dosage form such as an injection, fine granules, granules, capsules, tablets, etc. according to a conventional method using a pharmaceutically acceptable carrier or any auxiliary. It can be formulated. In this case, as an auxiliary agent, for example, an excipient, a binder, a disintegrant, a lubricant, a stabilizer, a flavoring / flavoring agent, and the like can be used.

  The pearl oyster extract or the ginger extract may be used by blending it in cosmetics, foods and drinks, etc., and can also be used as an external medicine.

  When the oyster extract or the ginger extract is blended in cosmetics, the blending amount can be appropriately adjusted, but is generally about 0.0001 to 10% by mass, preferably about 0.001 to 1% by mass. is there.

  The mussel extract or the ginger extract can be applied to the affected area as an external preparation, for example, as an emulsion, cream or gel ointment, or a patch. In this external preparation, in addition to mussel extract or ginger extract, oily raw materials, surfactants, inorganic fillers, thickeners, UV absorbers, antioxidants, preservatives, nutrients, colorants, fragrances, whitening agents, Various agents such as blood circulation promoters, local stimulants, anti-inflammatory agents, astringents, refreshing agents, skin roughening agents, antiperspirants, vitamins, hormones, or antihistamines can be appropriately blended depending on applications.

  The dose and frequency of administration of the mussel extract or ginger extract are not particularly limited, and are appropriately selected according to the conditions such as the purpose of prevention and / or treatment, the type of disease, the weight and age of the patient, and the severity of the disease. be able to. In general, the dose per day for an adult is about 0.1 to 1000 mg as the weight of an active ingredient mussel extract or ginger extract.

  The following examples further illustrate the present invention, but the present invention is not limited to the examples.

Manufacture example 1: Manufacture of methanol extract of mussel and shrimp Add 1 gram of dried mussel or 10 ml of methanol to the dried shrimp, extract at heating (70 ° C) or room temperature for 2 hours, filter off insolubles, concentrate under reduced pressure, and dry I let you. In the experiment, the extract was redissolved with dimethyl sulfoxide (DMSO) and adjusted to a concentration of 100 mg / ml.

Example 1: Action of various herbal extracts on aquaporin 3 mRNA expression in keratinocytes To the culture solution of keratinocyte strain DJM-1 cells, licorice, mussel, scorpion, takusha, touki and ginger methanol extracts (0.1 mg / ml) were added, After 24 hours of culturing, total cell RNA was extracted using TRIZOL reagent (Invitrogen). The amount of aquaporin 3 mRNA was measured by the Real time-PCR method and normalized based on the amount of GAPDH mRNA. In Real time-PCR, first, extracted total RNA was used as a starting material, and DNA was converted by reverse transcription using PrimeScript Reverse Transcriptase (Takara). SYBR using this as a mold Using Premix Ex Taq (Takara) and the following primers, amplification reaction was performed at (95 ° C. for 3 min) × 1 cycle, (95 ° C. for 15 sec, 60 ° C. for 1 min) × 40 cycles. The amount of mRNA was determined from the initial cycle number (threshold cycle: Ct value) in which a certain amount of PCR amplification product was detected, and quantified by comparison with the Ct value of GAPDH.

AQP3-Foward: 5'-TGCCTGGGGACCCTCATC-3 '(SEQ ID NO: 1)
AQP3-Reverse: 5'-GATCATATCCAAGTGTCC-3 '(SEQ ID NO: 2)
GAPDH- Forward: 5'-ACCATCTTCCAGGAGCGAGA-3 '(SEQ ID NO: 3)
GAPDH-Reverse: 5′-CAGTCTTCTGGGTGGCAGTG-3 ′ (SEQ ID NO: 4)

  The measurement result of the amount of aquaporin 3 mRNA is shown in FIG. In FIG. 1, the amount of aquaporin 3 mRNA was marked with a ratio of aquaporin 3 mRNA / GAPDH mRNA in the solvent administration group as 1. Means ± SD, n = 3, *: p <0.05 (Dunnett two-tailed test vs. control)

  As can be seen from the results in FIG. 1, significant increase in expression was observed in mussel, cypress and Tokyo, and significant suppression of expression was observed in Syria.

Example 2: Effect of various herbal extracts on aquaporin 3 protein expression in keratinocytes To the culture solution of keratinocyte strain DJM-1 cells, licorice, mussel, sardines, taksya, tokyo and ginger methanol extracts (0.1 mg / ml) were added. After 24 hours, cells were solubilized with RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% deoxycholate, 1% protease inhibitor cocktail; pH 7.5) Then, the supernatant obtained by centrifugation (14,000 × g, 4 ° C., 10 minutes) was used as a sample. The sample was separated on a 12% polyacrylamide gel and then transferred to a PVDF membrane. The transferred PVDF membrane was blocked with 5% skim milk, and primary antibody (anti-aquaporin 3 antibody, Chemicon or anti-β-actin antibody, Sigma, 4 ° C, 8 hours) and secondary antibody reaction (anti-rabbit antibody, Jackson labs) , Room temperature, 1 hour). Thereafter, the immune complex was chemiluminescent using ECL advance Western blotting analysis system (GE Healthcare Life Science) and detected with a bioimage analyzer (LAS-1000, Fuji Film).

  The measurement result of aquaporin 3 protein expression by Western blot is shown in FIG. As can be seen from the results in FIG. 2, only the cells treated with mussel showed an increase in the amount of aquaporin 3, and the amount of aquaporin 3 decreased in Sojutsu and Syougyo.

Example 3: Effect of mussel on the migration ability of keratinocytes To a culture solution of confluent culture of keratinocyte strain DJM-1 cells, a mussel extract (0.1 mg / ml) of kieselguhr or ginger was added, and after 24 hours of culture, Cells were scraped linearly once with the tip of a 200 μl pipette tip. Thereafter, 1% FBS was added to the culture solution, and further cultured for 24 hours. Photographs of the cells were taken under a microscope, and the width of the interval between the peeled cells was measured.

The measurement results are shown in FIG. The top of FIG. 3 shows a picture of the cell gap 24 hours after control, cells treated with mussel and cells treated with ginger. The lower graph in FIG. 3 indicates the result of this experiment as Means ± SD (n = 5). *: P <0.05, **: p <0.01 (Student's T-test vs. control)
As shown in FIG. 3, in the cells cultured in the presence of mussel, the gap width was significantly narrowed, and migration of keratinocytes was promoted. On the other hand, the width of the gap was significantly wider in the show, and migration was suppressed.

Example 4: Reduction of Aquaporin 3 mRNA Expression at Skin Inflammation Sites From each NCNga mouse that spontaneously developed dermatitis, three skins at each of the inflammation site and normal site were excised, and the amount of aquaporin 3 mRNA in these samples was semiquantitative PCR. We examined by the method. TRIZOL reagent (Invitrogen) was used for extraction of total RNA from skin tissue. Each extracted RNA sample was reverse transcribed and amplified using RNA PCR Kit Ver. 2 (Takara) to detect aquaporin 3 and HPRT mRNA as an internal standard. For reverse transcription, the oligo-dT primer contained in this kit was used and reacted at 42 ° C. for 60 minutes to form DNA. The primers described in SEQ ID NOs: 1 to 4 were used for the aquaporin 3 PCR reaction, and the primers shown below were used for HPRT. The amplified DNA fragment was electrophoresed on a 1.5% agarose gel and detected by ethidium bromide staining.

HPRT-Forward: 5'-GTTGGATACAGGCCAGACTTTGTTG-3 '(SEQ ID NO: 5)
HPRT-Reverse: 5'-GATTCAACTTGCGCTCATCTTAGGC-3 '(SEQ ID NO: 6)

  The results are shown in FIG. At any inflammation site, the amount of aquaporin 3 mRNA was reduced compared to normal skin, but there was no prominent effect on the amount of HPRT mRNA that is a marker for keratinocytes.

Example 5: Action of each fraction prepared from mussel methanol extract on aquaporin 3 mRNA expression Silicate mussel extract was adsorbed on a hydrophobic chromatography Diaion HP-20P column, and then acetone, 100% methanol, 50% methanol and water were added. The crude fraction was prepared by elution from a highly hydrophobic substance. Each fraction was added to the culture solution of the keratinocyte cell line DJM-1 at the above concentration, RNA was extracted 24 hours later, and the amount of aquaporin 3 mRNA was evaluated by a semi-quantitative RT-PCR method. In addition, the density | concentration of each fraction was set to the density | concentration in 0.1 mg / ml of mussel methanol extract computed based on the recovery rate. Semi-quantitative RT-PCR in this experiment was in accordance with the method of Example 4 above.

  The results are shown in FIG. The aquaporin 3 expression enhancing action mainly appeared in the 100% methanol fraction (partially acetone fraction), suggesting the involvement of a relatively hydrophobic substance.

Claims (1)

An aquaporin 3 expression enhancer for use as a wound healing promoter , comprising 100% methanol extract of mussel as an active ingredient.